ABSTRACT Millions of people worldwide suffer from neurological diseases such as Alzheimer's disease, stroke, and brain cancer. Advances in protein/gene profiling techniques and high throughput drug screening technologies have spawned many new drug candidates. However, the blood-brain barrier (BBB) has impeded the development and clinical realization of this new generation of neurotherapeutics by restricting the brain uptake of most small molecule therapeutics, and prohibiting brain uptake of protein- and gene-based medicines. A promising noninvasive brain delivery strategy takes advantage of endogenous BBB transport mechanisms as a means to shuttle drug cargo from the blood to the brain. Such receptor-mediated transport systems can be targeted using the exquisite specificity of antibodies that are in turn linked to a drug payload that can include small molecules, proteins, or DNA therapeutics. After binding to the receptor on the blood side, the antibody- drug conjugate acts as an artificial substrate for the transporter and is transcytosed from the blood, across the BBB, and into the brain. Current approaches have yielded limited brain uptake because the targeted transporters are ubiquitously expressed, and the antibody targeting reagents have a low BBB permeability. Therefore, this proposal is focused on the identification and validation of novel delivery vectors and their cognate BBB transporters that can mediate improved transport efficiency. Rather than deploying traditional mammalian antibody technology, we describe a new strategy that employs lamprey antibodies known as Variable Lymphocyte Receptors (VLRs) to target the BBB. Lampreys and humans last shared a common ancestor >500 million years ago, and due to this tremendous evolutionary divergence, even highly conserved mammalian proteins and carbohydrates are immunogenic in lampreys. By leveraging these unique aspects of the lamprey immune system with innovative screening technologies, we anticipate that the proposed research will provide new BBB-targeting VLRs capable of trafficking into the brain. To achieve these goals, lampreys were immunized with mouse brain microvessel plasma membrane preparations, and staining with the resultant polyclonal antiserum demonstrated that VLRs clearly recognize the in vivo BBB and bind to multiple unique glycan structures. The lymphocyte cDNA of immunized lampreys was then used to create a yeast display library consisting of millions of VLRs that will be screened using an innovative screening approach to select BBB-binding and trafficking monoclonal VLRs. These brain-targeting VLRs will be validated both by pharmacokinetic profiling and their capacity to elicit a pharmacologically- relevant response in a murine stroke model. Finally, the BBB-targeting VLRs will be employed to identify the cognate BBB transporter and any associated glycoforms. Those VLRs exhibiting significant and specific brain uptake would represent new, noninvasive brain drug delivery vectors that could be powerful in the treatment of debilitating neurological disease.